The present invention relates generally to an integrated miniaturized fluidic system fabricated using microelectromechanical systems (MEMS) technology, particularly to an integrated monolithic microfabricated dispensing nozzle capable of dispensing fluids in the form of droplets or as an electrospray of the fluid.
New trends in drug discovery and development are creating new demands on analytical techniques. For example, combinatorial chemistry is often employed to discover new lead compounds, or to create variations of a lead compound. Combinatorial chemistry techniques can generate thousands of compounds (combinatorial libraries) in a relatively short time (on the order of days to weeks). Testing such a large number of compounds for biological activity in a timely and efficient manner requires high-throughput screening methods which allow rapid evaluation of the characteristics of each candidate compound.
The compounds in combinatorial libraries are often tested simultaneously against a molecular target. For example, an enzyme assay employing a calorimetric measurement may be run in a 96-well plate. An aliquot of enzyme in each well is combined with tens of compounds. An effective enzyme inhibitor will prevent development of color due to the normal enzyme reaction, allowing for rapid spectroscopic (or visual) evaluation of assay results. If ten compounds are present in each well, 960 compounds can be screened in the entire plate, and one hundred thousand compounds can be screened in 105 plates, allowing for rapid and automated biological screening of the compounds.
The quality of the combinatorial library and the compounds contained therein is used to assess the validity of the biological screening data. Confirmation that the correct molecular weight is identified for each compound or a statistically relevant number of compounds along with a measure of compound purity are two important measures of the quality of a combinatorial library. Compounds can be analytically characterized by removing a portion of solution from each well and injecting the contents into a separation device such as liquid chromatography or capillary electrophoresis instrument coupled to a mass spectrometer. Assuming that such a method would take approximately 5 minutes per analysis, it would require over a month to analyze the contents of 105 96-well plates, assuming the method was fully automated and operating 24 hours a day. Even larger well-plates containing 384 and 1536 wells are being integrated into the screening of new chemical entities imposing even greater time constraints on the analytical characterization of these libraries.
Recent technological developments in combinatorial chemistry, molecular biology, and new microchip chemical devices have created the need for new types of dispensing devices. Applications in combinatorial chemistry require robust sample delivery systems that are chemically inert and distribute less than microliter amounts of liquid in high-density formats. The systems need to be highly reproducible and have overall quick dispensing times. Current dispensing technology utilizes serial injection schemes. The use of serial dispensers will be inherently limited due to their slow overall distribution times as the move to high-density formats progresses. For example, for combinatorial chemistry applications, to synthesize a library of 1 million discrete compounds, each composed of 4 monomers, a total of 4xc3x97106 dispensing steps would be required. If each dispensing step required 3 seconds (considering dispense time, rinsing, and, location positioning), the total time to dispense all of the reagents would be 12xc3x97106 seconds, or 3333 hours, or 139 days. Thus, for high-density formats, dispensing must be conducted in parallel. In order for parallel dispensing to work in high-density formats, the dispensing device must be small enough to allow all dispensing units to be simultaneously positioned within a corresponding receiving well. This requires the dispenser to be relatively small. As high density formats reach greater than 10,000 wells, dispensing devices will need to be spaced within 100 xcexcm or less. In addition, in order for the dispenser to be practical, the device must dispense small quantities of liquid (10xe2x88x929 to 10xe2x88x9212 L), and only require small volumes to operate.
Piezoelectric dispensing units have also been used for dispensing small amounts of liquid for microdevices. However, piezoelectric dispensers suffer from several problems. Currently, the closest spacing of individual dispensers is 330 xcexcm in an array of four. Due to the current piezoelectric design and fabrication, the number of dispensers that can be positioned adjacent to one another is limited because of downstream device features. Additionally, sample requirements may be quite high even though the dispensing volume is small.
Enormous amounts of genetic sequence data are being generated through new DNA sequencing methods. This wealth of new information is generating new insights into the mechanism of disease processes. In particular, the burgeoning field of genomics has allowed rapid identification of new targets for drug discovery. Determination of genetic variations between individuals has opened up the possibility of targeting drugs to individuals based on the individual""s particular genetic profile. Testing for cytotoxicity, specificity, and other pharmaceutical La characteristics could be carried out in high-throughput assays instead of expensive animal testing and clinical trials. Detailed characterization of a potential drug or lead compound early in the drug development process thus has the potential for significant savings both in time and expense.
Development of viable screening methods for these new targets will often depend on the availability of rapid separation and analysis techniques for analyzing the results of assays. For example, an assay for potential toxic metabolites of a candidate drug would need to identify both the candidate drug and the metabolites of that candidate. An assay for specificity would need to identify compounds that bind differentially to two molecular targets such as a viral protease and a mammalian protease.
It would, therefore, be advantageous to provide a method for efficient proteomic screening in order to obtain the pharmacokinetic profile of a drug early in the evaluation process. An understanding of how a new compound is absorbed in the body and how it is metabolized can enable prediction of the likelihood for an increased therapeutic effect or lack thereof.
Given the enormous number of new compounds that are being generated daily, an improved system for identifying molecules of potential therapeutic value for drug discovery is also critically needed. Accordingly, there is a critical need for high-throughput screening and identification of compound-target reactions in order to identify potential drug candidates.
Liquid chromatography (LC) is a well-established analytical method for separating components of a fluid for subsequent analysis and/or identification. Traditionally, liquid chromatography utilizes a separation column, such as a cylindrical tube with dimensions 4.6 mm inner diameter by 25 cm length, filled with tightly packed particles of 5 xcexcm diameter. More recently, particles of 3 xcexcm diameter are being used in shorter length columns. The small particle size provides a large surface area that can be modified with various chemistries creating a stationary phase. A liquid eluent is pumped through the LC column at an optimized flow rate based on the column dimensions and particle size. This liquid eluent is referred to as the mobile phase. A volume of sample is injected into the mobile phase prior to the LC column. The analytes in the sample interact with the stationary phase based on the partition coefficients for each of the analytes. The partition coefficient is defined as the ratio of the time an analyte spends interacting with the stationary phase to the time spent interacting with the mobile phase. The longer an analyte interacts with the stationary phase, the higher the partition coefficient and the longer the analyte is retained on the LC column. The diffusion rate for an analyte through a mobile phase (mobile-phase mass transfer) also affects the partition coefficient. The mobile-phase mass transfer can be rate limiting in the performance of the separation column when it is greater than 2 xcexcm (Knox, J. H. J. J. Chromatogr. Sci. 18:453-461 (1980)). Increases in chromatographic separation are achieved when using a smaller particle size as the stationary phase support.
The purpose of the LC column is to separate analytes such that a unique response for each analyte from a chosen detector can be acquired for a quantitative or qualitative measurement. The ability of a LC column to generate a separation is determined by the dimensions of the column and the particle size supporting the stationary phase. A measure of the ability of LC columns to separate a given analyte is referred to as the theoretical plate number N. The retention time of an analyte can be adjusted by varying the mobile phase composition and the partition coefficient for an analyte. Experimentation and a fundamental understanding of the partition coefficient for a given analyte determine which stationary phase is chosen.
To increase the throughput of LC analyses requires a reduction in the dimensions of the LC column and the stationary phase particle dimensions. Reducing the length of the LC column from 25 cm to 5 cm will result in a factor of 5 decrease in the retention time for an analyte. At the same time, the theoretical plates are reduced 5-fold. To maintain the theoretical plates of a 25 cm length column packed with 5 xcexcm particles, a 5 cm column would need to be packed with 1 xcexcm particles. However, the use of such small particles results in many technical challenges.
One of these technical challenges is the backpressure resulting from pushing the mobile phase through each of these columns. The backpressure is a measure of the pressure generated in a separation column due to pumping a mobile phase at a given flow rate through the LC column. For example, the typical backpressure of a 4.6 mm inner diameter by 25 cm length column packed with 5 xcexcm particles generates a backpressure of 100 bar at a flow rate of 1.0 mL/min. A 5 cm column packed with 1 xcexcm particles generates a back pressure 5 times greater than a 25 cm column packed with 5 xcexcm particles. Most commercially available LC pumps are limited to operating pressures less than 400 bar and thus using an LC column with these small particles is not feasible.
Detection of analytes separated on an LC column has traditionally been accomplished by use of spectroscopic detectors. Spectroscopic detectors rely on a change in refractive index, ultraviolet and/or visible light absorption, or fluorescence after excitation with a suitable wavelength to detect the separated components. Additionally, the effluent from an LC column may be nebulized to generate an aerosol which is sprayed into a chamber to measure the light scattering properties of the analytes eluting from the column. Alternatively, the separated components may be passed from the liquid chromatography column into other types of analytical instruments for analysis. The volume from the LC column to the detector is minimized in order to maintain the separation efficiency and analysis sensitivity. All system volume not directly resulting from the separation column is referred to as the dead volume or extra-column volume.
The miniaturization of liquid separation techniques to the nano-scale involves small column internal diameters ( less than 100 xcexcm i.d.) and low mobile phase flow rates ( less than 300 nL/min). Currently, techniques such as capillary zone electrophoresis (CZE), nano-LC, open tubular liquid chromatography (OTLC), and capillary electrochromatography (CEC) offer numerous advantages over conventional scale high performance liquid chromatography (HPLC). These advantages include higher separation efficiencies, high-speed separations, analysis of low volume samples, and the coupling of 2-dimensional techniques. One challenge to using miniaturized separation techniques is detection of the small peak volumes and a limited number of detectors that can accommodate these small volumes. However, coupling of low flow rate liquid separation techniques to electrospray mass spectrometry results in a combination of techniques that are well suited as demonstrated in J. N. Alexander IV, et al., Rapid Commun. Mass Spectrom. 12:1187-91 (1998). The process of electrospray at flow rates on the order of nanoliters per minute has been referred to as xe2x80x9cnanoelectrosprayxe2x80x9d.
Capillary electrophoresis is a technique that utilizes the electrophoretic nature of molecules and/or the electroosmotic flow of fluids in small capillary tubes to separate components of a fluid. Typically, a fused silica capillary of 100 xcexcm inner diameter or less is filled with a buffer solution containing an electrolyte. Each end of the capillary is placed in a separate fluidic reservoir containing a buffer electrolyte. A potential voltage is placed in one of the buffer reservoirs and a second potential voltage is placed in the other buffer reservoir. Positively and negatively charged species will migrate in opposite directions through the capillary under the influence of the electric field established by the two potential voltages applied to the buffer reservoirs. Electroosmotic flow is defined as the fluid flow along the walls of a capillary due to the migration of charged species from the buffer solution under the influence of the applied electric field. Some molecules exist as charged species when in solution and will migrate through the capillary based on the charge-to-mass ratio of the molecular species. This migration is defined as electrophoretic mobility. The electroosmotic flow and the electrophoretic mobility of each component of a fluid determine the overall migration for each fluidic component. The fluid flow profile resulting from electroosmotic flow is flat due to the reduction in frictional drag along the walls of the separation channel. This results in improved separation efficiency compared to liquid chromatography where the flow profile is parabolic resulting from pressure driven flow.
Capillary electrochromatography is a hybrid technique that utilizes the electrically driven flow characteristics of electrophoretic separation methods within capillary columns packed with a solid stationary phase typical of liquid chromatography. It couples the separation power of reversed-phase liquid chromatography with the high efficiencies of capillary electrophoresis. Higher efficiencies are obtainable for capillary electrochromatography separations over liquid chromatography, because the flow profile resulting from electroosmotic flow is flat due to the reduction in frictional drag along the walls of the separation channel when compared to the parabolic flow profile resulting from pressure driven flows. Furthermore, smaller particle sizes can be used in capillary electrochromatography than in liquid chromatography, because no backpressure is generated by electroosmotic flow. In contrast to electrophoresis, capillary electrochromatography is capable of separating neutral molecules due to analyte partitioning between the stationary and mobile phases of the column particles using a liquid chromatography separation mechanism.
Microchip-based separation devices have been developed for rapid analysis of large numbers of samples. Compared to other conventional separation devices, these microchip-based separation devices have higher sample throughput, reduced sample and reagent consumption, and reduced chemical waste. The liquid flow rates for microchip-based separation devices range from approximately 1-300 nanoliters (nL) per minute for most applications. Examples of microchip-based separation devices include those for capillary electrophoresis (xe2x80x9cCExe2x80x9d), capillary electrochromatography (xe2x80x9cCECxe2x80x9d) and high-performance liquid chromatography (xe2x80x9cHPLCxe2x80x9d) include Harrison et al., Science 261:859-97 (1993); Jacobson et al., Anal. Chem. 66:1114-18 (1994), Jacobson et al., Anal. Chem. 66:2369-73 (1994), Kutter et al., Anal. Chem. 69:5165-71 (1997) and He et al., Anal. Chem. 70:3790-97 (1998). Such separation devices are capable of fast analyses and provide improved precision and reliability compared to other conventional analytical instruments.
The work of He et al., Anal. Chem. 70:3790-97 (1998) demonstrates some of the types of structures that can be fabricated in a glass substrate. This work shows that co-located monolithic support structures (or posts) can be etched reproducibly in a glass substrate using reactive ion etching (RIE) techniques. Currently, anisotropic RIE techniques for glass substrates are limited to etching features that are 20 xcexcm or less in depth. This work shows rectangular 5 xcexcm by 5 xcexcm width by 10 xcexcm in depth posts and stated that deeper structures were difficult to achieve. The posts are also separated by 1.5 xcexcm. The posts supports the stationary phase just as with the particles in LC and CEC columns. An advantage to the posts over conventional LC and CEC is that the stationary phase support structures are monolithic with the substrate and therefore, immobile.
He et. al., also describes the importance of maintaining a constant cross-sectional area across the entire length of the separation channel. Large variations in the cross-sectional area can create pressure drops in pressure driven flow systems. In electrokinetically driven flow systems, large variations in the cross-sectional area along the length of a separation channel can create flow restrictions that result in bubble formation in the separation channel. Since the fluid flowing through the separation channel functions as the source and carrier of the mobile solvated ions, formation of a bubble in a separation channel will result in the disruption of the electroosmotic flow.
Electrospray ionization provides for the atmospheric pressure ionization of a liquid sample. The electrospray process creates highly-charged droplets that, under evaporation, create ions representative of the species contained in the solution. An ion-sampling orifice of a mass spectrometer may be used to sample these gas phase ions for mass analysis. A schematic of an electrospray system 50 is shown in FIG. 1A. An electrospray is produced when a sufficient electrical potential difference Vspray is applied between a conductive or partly conductive fluid exiting a capillary 52 and an extracting electrode 54 to generate a concentration of electric field lines emanating from the tip or end of a capillary 56. When a positive voltage Vspray is applied to the tip of the capillary relative to an extracting electrode, such as one provided at the ion-sampling orifice of a mass spectrometer, the electric field causes positively-charged ions in the fluid to migrate to the surface of the fluid at the tip of the capillary. When a negative voltage Vspray is applied to the tip of the capillary relative to an extracting electrode, such as one provided at the ion-sampling orifice to the mass spectrometer, the electric field causes negatively-charged ions in the fluid to migrate to the surface of the fluid at the tip of the capillary.
When the repulsion force of the solvated ions on the surface of the fluid exceeds the surface tension of the fluid being electrosprayed, a volume of the fluid is pulled into the shape of a cone, known as a Taylor cone 58, which extends from the tip of the capillary 56. A liquid jet 60 extends from the tip of the Taylor cone and becomes unstable and generates charged-droplets 62. These small charged droplets are drawn toward the extracting electrode 54. The small droplets are highly-charged and solvent evaporation from the droplets results in the excess charge in the droplet residing on the analyte molecules in the electrosprayed fluid. The charged molecules or ions are drawn through the ion-sampling orifice of the mass spectrometer for mass analysis. This phenomenon has been described, for example, by Dole et al., Chem. Phys. 49:2240 (1968) and Yamashita et al., J. Phys. Chem. 88:4451 (1984). The potential voltage required to initiate an electrospray is dependent on the surface tension of the solution as described by, for example, Smith, IEEE Trans. Ind. Appl. 1986, IA-22:527-35 (1986). Typically, the electric field is on the order of approximately 106 V/m. The physical size of the capillary and the fluid surface tension determines the density of electric field lines necessary to initiate electrospray.
When the repulsion force of the solvated ions is not sufficient to overcome the surface tension of the fluid exiting the tip of the capillary, large poorly charged droplets are formed as shown in FIG. 1B. Fluid droplets 64 are produced when the electrical potential difference Vdroplet applied between a conductive or partly conductive fluid exiting a capillary 52 and an electrode is not sufficient to overcome the fluid surface tension to form a Taylor cone.
Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Applications, edited by R. B. Cole, ISBN 0-471-14564-5, John Wiley and Sons, Inc., New York summarizes much of the fundamental studies of electrospray. Several mathematical models have been generated to explain the principals governing electrospray. Equation 1 defines the electric field Ec at the tip of a capillary of radius rc with an applied voltage Vc at a distance d from a counter electrode held at ground potential:                               E          c                =                              2            ⁢                          V              c                                                          r              c                        ⁢            ln            ⁢                          xe2x80x83                        ⁢                          (                              4                ⁢                                  d                  /                                      r                    c                                                              )                                                          (        1        )            
The electric field Eon required for the formation of a Taylor cone and liquid jet of a fluid flowing to the tip of this capillary is approximated as:                               E          on                ≈                              (                                          2                ⁢                                  xe2x80x83                                ⁢                γ                ⁢                                  xe2x80x83                                ⁢                cos                ⁢                                  xe2x80x83                                ⁢                θ                                                              ϵ                  o                                ⁢                                  r                  c                                                      )                                1            /            2                                              (        2        )            
where xcex3 is the surface tension of the fluid, xcex8 is the half-angle of the Taylor cone and xcex50 is the permittivity of vacuum. Equation 3 is derived by combining equations 1 and 2 and approximates the onset voltage Von required to initiate an electrospray of a fluid from a capillary:                               V          on                ≈                                            (                                                                    r                    c                                    ⁢                  γ                  ⁢                                      xe2x80x83                                    ⁢                  cos                  ⁢                                      xe2x80x83                                    ⁢                  θ                                                  2                  ⁢                                      xe2x80x83                                    ⁢                                      ϵ                    0                                                              )                                      1              /              2                                ⁢          ln          ⁢                      xe2x80x83                    ⁢                      (                          4              ⁢                              d                /                                  r                  c                                                      )                                              (        3        )            
The graph of FIG. 1C shows curves for onset voltages of 500, 750 and 1000 V as related to surface tension of a fluid undergoing electrospray from the tip of a capillary with a given outer diameter. The distance of the capillary tip from the counter-electrode was fixed at 2 mm. Combinations of fluid surface tension and capillary diameters that fall below the curves will generate a stable electrospray using a given onset voltage. As can be seen by examination of equation 3, the required onset voltage is more dependent on the capillary radius than the distance from the counter-electrode.
It would be desirable to define an electrospray device that could form a stable electrospray of all fluids commonly used in CE, CEC, and LC. The surface tension of solvents commonly used as the mobile phase for these separations range from 100% aqueous (xcex3=0.073 N/m) to 100% methanol (xcex3=0.0226 N/m). FIG. 1C indicates that as the surface tension of the electrospray fluid increases, a higher onset voltage is required to initiate an electrospray for a fixed capillary diameter. As an example, a capillary with a tip diameter of 14 xcexcm is required to electrospray 100% aqueous solutions with an onset voltage of 1000 V. The work of M. S. Wilm et al., Int. J. Mass Spectrom. Ion Processes 136:167-80 (1994), first demonstrates nanoelectrospray from a fused-silica capillary pulled to an outer diameter of 5 xcexcm at a flow rate of 25 nL/min. Specifically, a nanoelectrospray at 25 nL/min was achieved from a 2 xcexcm inner diameter and 5 xcexcm outer diameter pulled fused-silica capillary with 600-700 V at a distance of 1-2 mm from the ion-sampling orifice of an electrospray equipped mass spectrometer.
Electrospray in front of an ion-sampling orifice of an API mass spectrometer produces a quantitative response from the mass spectrometer detector due to the analyte molecules present in the liquid flowing from the capillary. One advantage of electrospray is that the response for an analyte measured by the mass spectrometer detector is dependent on the concentration of the analyte in the fluid and independent of the fluid flow rate. The response of an analyte in solution at a given concentration would be comparable using electrospray combined with mass spectrometry at a flow rate of 100 xcexcL/min compared to a flow rate of 100 nL/min. D. C. Gale et al., Rapid Commun. Mass Spectrom. 7:1017 (1993) demonstrate that higher electrospray sensitivity is achieved at lower flow rates due to increased analyte ionization efficiency.
Attempts have been made to manufacture an electrospray device for microchip-based separations. Ramsey et al., Anal. Chem. 69:1174-78 (1997) describes a microchip-based separations device coupled with an electrospray mass spectrometer. Previous work from this research group including Jacobson et al., Anal. Chem. 66:1114-18 (1994) and Jacobson et al., Anal. Chem. 66:2369-73 (1994) demonstrate impressive separations using on-chip fluorescence detection. This more recent work demonstrates nanoelectrospray at 90 nL/min from the edge of a planar glass microchip. The microchip-based separation channel has dimensions of 10 xcexcm deep, 60 xcexcm wide, and 33 mm in length. Electroosmotic flow is used to generate fluid flow at 90 nL/min. Application of 4,800 V to the fluid exiting the separation channel on the edge of the microchip at a distance of 3-5 mm from the ion-sampling orifice of an API mass spectrometer generates an electrospray. Approximately 12 nL of the sample fluid collects at the edge of the microchip before the formation of a Taylor cone and stable nanoelectrospray from the edge of the microchip. The volume of this microchip-based separation channel is 19.8 nL. Nanoelectrospray from the edge of this microchip device after capillary electrophoresis or capillary electrochromatography separation is rendered impractical since this system has a dead-volume approaching 60% of the column (channel) volume. Furthermore, because this device provides a flat surface, and, thus, a relatively small amount of physical asperity for the formation of the electrospray, the device requires an impractically high voltage to overcome the fluid surface tension to initiate an electrospray.
Xue, Q. et al., Anal. Chem. 69:426-30 (1997) also describes a stable nanoelectrospray from the edge of a planar glass microchip with a closed channel 25 xcexcm deep, 60 xcexcm wide, and 35-50 mm in length. An electrospray is formed by applying 4,200 V to the fluid exiting the separation channel on the edge of the microchip at a distance of 3-8 mm from the ion-sampling orifice of an API mass spectrometer. A syringe pump is utilized to deliver the sample fluid to the glass microchip at a flow rate of 100 to 200 nL/min. The edge of the glass microchip is treated with a hydrophobic coating to alleviate some of the difficulties associated with nanoelectrospray from a flat surface that slightly improves the stability of the nanoelectrospray. Nevertheless, the volume of the Taylor cone on the edge of the microchip is too large relative to the volume of the separation channel, making this method of electrospray directly from the edge of a microchip impracticable when combined with a chromatographic separation device.
T. D. Lee et. al., 1997 International Conference on Solid-State Sensors and Actuators Chicago, pp. 927-30 (Jun. 16-19, 1997) describes a multi-step process to generate a nozzle on the edge of a silicon microchip 1-3 xcexcm in diameter or width and 40 xcexcm in length and applying 4,000 V to the entire microchip at a distance of 0.25-0.4 mm from the ion-sampling orifice of an API mass spectrometer. Because a relatively high voltage is required to form an electrospray with the nozzle positioned in very close proximity to the mass spectrometer ion-sampling orifice, this device produces an inefficient electrospray that does not allow for sufficient droplet evaporation before the ions enter the orifice. The extension of the nozzle from the edge of the microchip also exposes the nozzle to accidental breakage. More recently, T. D. Lee et.al., in 1999 Twelfth IEEE International Micro Electro Mechanical Systems Conference (Jan. 17-21, 1999), presented this same concept where the electrospray component was fabricated to extend 2.5 mm beyond the edge of the microchip to overcome this phenomenon of poor electric field control within the proximity of a surface.
In all of the above-described devices, generating an electrospray from the edge of a microchip is a poorly controlled process. These devices do not define a nozzle and an electric field around the nozzle that is required to produce a stable and highly reproducible electrospray. In another embodiment, small segments of fused-silica capillaries are separately and individually attached to the chip""s edge. This process is inherently cost-inefficient and unreliable, imposes space constraints in chip design, and is therefore unsuitable for manufacturing.
Thus, it is also desirable to provide an electrospray device with controllable spraying and a method for producing such a device that is easily reproducible and manufacturable in high volumes.
U.S. Pat. No. 5,501,893 to Laermer et. al., reports a method of anisotropic plasma etching of silicon (Bosch process) that provides a method of producing deep vertical structures that is easily reproducible and controllable. This method of anisotropic plasma etching of silicon incorporates a two step process. Step one is an anisotropic etch step using a reactive ion etching (RIE) gas plasma of sulfur hexafluoride (SF6). Step two is a passivation step that deposits a polymer on the vertical surfaces of the silicon substrate. This polymerizing step provides an etch stop on the vertical surface that was exposed in step one. This two step cycle of etch and passivation is repeated until the depth of the desired structure is achieved. This method of anisotropic plasma etching provides etch rates over 3 xcexcm/min of silicon depending on the size of the feature being etched. The process also provides selectivity to etching silicon versus silicon dioxide or resist of greater than 100:1 which is important when deep silicon structures are desired. Laermer et. al., in 1999 Twelfth IEEE International Micro Electro Mechanical Systems Conference (Jan. 17-21, 1999), reported improvements to the Bosch process. These improvements include silicon etch rates approaching 10 xcexcm/min, selectivity exceeding 300:1 to silicon dioxide masks, and more uniform etch rates for features that vary in size.
The electrical properties of silicon and silicon-based materials are well characterized. The use of silicon dioxide and silicon nitride layers grown or deposited on the surfaces of a silicon substrate are well known to provide electrical insulating properties. Silicon dioxide layers may be grown thermally in an oven to a desired thickness. Silicon nitride can be deposited using low pressure chemical vapor deposition (LPCVD). Metals may be further vapor deposited on these surfaces to provide for application of a potential voltage on the surface of the device. Both silicon dioxide and silicon nitride function as electrical insulators allowing the application of a potential voltage to the substrate that is different than that applied to the surface of the device. An important feature of a silicon nitride layer is that it provides a moisture barrier between the silicon substrate, silicon dioxide and any fluid sample that comes in contact with the device. Silicon nitride prevents water and ions from diffusing through the silicon dioxide layer to the silicon substrate which may cause an electrical breakdown between the fluid and the silicon substrate. Additional layers of silicon dioxide, metals and other materials may further be deposited on the silicon nitride layer to provide chemical functionality to silicon-based devices.
The present invention is directed to overcoming the deficiencies in prior electrospray systems.
The present invention relates to an electrospray device which comprises a substrate having an injection surface and an ejection surface opposing the injection surface with the substrate being an integral monolith. An entrance orifice is positioned on the injection surface, while an exit orifice is on the ejection surface. A channel extends between the entrance orifice and the exit orifice. A recess surrounds the exit orifice and is positioned between the injection surface and the ejection surface. The electrospray device has voltage application system consisting essentially of a first electrode attached to the substrate to impart a first potential to the substrate and a second electrode to impart a second potential, where the first and the second electrodes are positioned to define an electric field surrounding the exit orifice. This device can be used in conjunction with systems for processing droplet/sprays, methods of generating an electrospray, a method of mass spectrometeric analysis, and a method of liquid chromatographic analysis.
Another aspect of the present invention is directed to an electrospray device which includes a capillary tube having a passage for conducting fluids through the capillary tube and connecting an entrance orifice and an exit orifice, a first electrode circumscribing the capillary tube proximate the exit orifice, and a second electrode to impart a second potential. The first and the second electrodes are positioned to define an electric field surrounding the exit orifice.
Another aspect of the present invention relates to a method of producing an electrospray device which includes providing a substrate having opposed first and second surfaces, each coated with a photoresist. The photoresist on the first surface is exposed to an optical image to form a pattern is the form of a spot on the first surface. The photoresist on the first surface where the pattern is removed to form a hole in the photoresist. Material is removed from the substrate coincident with the hole in the photoresist on the first surface to form a channel extending through the photoresist on the first surface and through the substrate up to the photoresist on the second surface. The photoresist on the second surface is exposed to an image to form an annular pattern circumscribing an extension of the channel through the photoresist on the second surface. The photoresist on the second surface having the annular pattern is then removed, and, next, the material from the substrate coincident with the removed annular pattern in the phototresist on the second surface is removed to form an annular recess extending partially into the substrate. All coatings from the first and second surfaces of the substrate are removed to form the electrospray device.
Another aspect of the present invention relates to a method of producing an electrospray device. This method includes providing a substrate having opposed first and second surfaces, each coated with a photoresist. The photoresist is exposed on the first surface to an image to form a pattern in the form of at least 3 substantially aligned spots on the first surface. The photoresist on the first surface is removed where the pattern is to form 3 holes in the photoresist corresponding to where the spots in the photoresist were. Material from the substrate coincident with the removed pattern in the photoresist on the first surface is then removed to form a central channel aligned with and between two outer channels. The channels extend through the photoresist on the first surface and into the substrate. The central channel has a diameter which is less than that of the outer channels such that the central channel extends farther from the second surface of the substrate than the outer channels which extend up to the photoresist on the the second surface. The photoresist on the second surface is exposed to an image which forms an annular pattern circumscribing a spot, where the spot is coincident with an extension of the central channel through the photoresist on the second surface and a portion of the substrate. The photoresist on the second surface is removed where the annular pattern circumscribing the spot is. Material from the substrate coincident with the removed pattern in the photoresist on the second surface is then removed. This forms an annular recess extending partially into the substrate and circumscribing the central channel which extends through the substrate and the photoresist on the first and second surfaces. All coatings from the first and second surfaces of the substrate are then removed. All surfaces of the substrate are then coated with an insulating material to form the electrospray device.
Another aspect of the present invention relates to a method of forming a liquid separation device. This method involves providing a substrate having opposed first and second surfaces, each coated with a photoresist. The photoresist is exposed on the first surface to an image to form a pattern in the form of a plurality of spots on the first surface. The photoresist on the first surface where the pattern is is removed to form a plurality of holes in the photoresist corresponding to where the spots in the photoresist were. Material from the substrate coincident with where the pattern in the photoresist on the first surface has been removed is then removed. This forms a large reservoir proximate a first end of the substrate and a plurality of smaller holes closer to a second opposite end of the substrate than the reservoir. The reservoir and holes extend through the photoresist on the first surface and partially into the substrate. The smaller holes and the surfaces of the reservoir are filled with a coating, and a further photoresist layer is applied over the coating on the surfaces of the reservoir, the filled holes, and the photoresist on the first surface. The further photoresist is exposed to an image to form a pattern in the form of spots, with one spot coincident with what was the reservoir and the other spot being closer to the second end of the substrate than the filled holes. The further photoresist is removed where the pattern is to form holes corresponding to where the spots in the photoresist were. Material is removed from the substrate coincident with where the pattern in the further photoresist has been removed to form a pair of channels. A first channel extends through what was the reservoir up to the photoresist on the second surface. A second channel extends through the substrate up to the photoresist on the second surface at a location closer to the second end of the substrate than the filled holes. All coatings from the first and second surfaces of the substrate are removed, and all surfaces of the substrate are coated with an insulating material to form the liquid separation device.
Another aspect of the present invention relates to a system for processing droplets/sprays of fluid which includes an electrospray device. The electrospay device contains a substrate having an injection surface and an ejection surface opposing the injection surface. The substrate comprises an entrance orifice on the injection surface, an exit orifice on the ejection surface, a channel extending between the entrance orifice and the exit orifice, and a recess extending into the ejection surface and surrounding the exit orifice. The system further includes a device to provide fluid to the electrospray device which includes a fluid passage, a fluid reservoir in fluid communication with the fluid passage, a fluid inlet to direct fluid entering the device into the fluid reservoir, and a fluid outlet to direct fluid from the fluid passage to the entrance orifice of the electrospray device. The cross-sectional area of the entrance orifice of the electrospray device is equal to or less than the cross-sectional area of the fluid passage.
The present invention achieves a significant advantage in terms of high-throughput distribution and apportionment of massively parallel channels of discrete chemical entities in a well-controlled, reproducible method. An array of dispensing nozzles is disclosed for application in inkjet printing. When combined with a miniaturized liquid chromatography system and method, the present invention achieves a significant advantage in comparison to a conventional system.
The present invention insulates a fluid introduced to the electrospray device from the silicon substrate of the device. This insulation is in the form of silicon dioxide and silicon nitride layers contained on the surfaces of the electrospray device. These insulating layers allow for independent application of a voltage to the fluid introduced to the electrospray device and the voltage applied to the substrate. The independent voltage application to the fluid and substrate allow for control of the electric field around the exit orifice of the nozzle on the ejection surface of the electrospray device independent of the need for any additional electrodes or voltages. This, combined with the dimensions of the nozzle formed from the ejection surface of the electrospray device and the fluid surface tension, determine the electric field and voltages required for the formation of droplets or an electrospray from this invention.
The electrospray device of the present invention can be integrated with microchip-based devices having atmospheric pressure ionization mass spectrometry (API-MS) instruments. By generating an electric field at the tip of a nozzle, which exists in a planar or near planar geometry with the ejection surface of a substrate, fluid droplets and an electrospray exiting the nozzle on the ejection surface are efficiently generated. When a nozzle exists in this co-planar or near planar geometry, the electric field lines emanating from the tip of the nozzle will not be enhanced if the electric field around the nozzle is not defined and controlled.
Control of the electric field at the tip of a nozzle formed from a substrate for the efficient formation of droplets and electrospray from a microchip is an important aspect of the present invention. This was determined using a fused-silica capillary 52 pulled to an outer diameter of approximately 20 xcexcm and inserted through a ring electrode 70 with a 1 mm diameter as shown in FIG. 2. FIG. 2A shows a plan view of the capillary/ring electrode experiment. FIG. 2B shows a cross-sectional view of the capillary/ring electrode experiment. The capillary tip 56 is inserted up to 5 mm through the ring electrode 70 in front of an ion-sampling orifice 54 of a mass spectrometer equipped with an electrospray ion source. A voltage of 700 V is applied to an aqueous fluid Vfluid flowing to the capillary tip at a flow rate of 50 nL/min. The ring electrode 70 is mounted on an XYZ stage to allow the ring electrode to be moved slowly forward to the point at which the capillary tip 56 is co-planar with the ring electrode 70 as shown in FIGS. 2C and 2D. The voltage applied to the ring electrode Vring is variable. The voltage applied to the ion-sampling orifice 54 is 80 V. When the fluid voltage and the ring electrode voltage are maintained at 700 V in the co-planar geometry, the electrospray is disrupted and no longer forms an electrospray. Depending on the Vfluid/Vring ratio for a fixed distance from a counter electrode 54, fluidic droplets can be controllably dispensed from the capillary tip as shown in FIG. 2C. In this case, minimally-charged, larger droplets are formed with the droplet diameter dependent on the electric field established by the Vfluid/Vring ratio, fluid surface tension, fluid conductivity, capillary tip diameter and distance from a counter electrode. Application of a voltage of 0 V to the ring electrode 70 results in the formation of a stable electrospray once again as shown in FIG. 2D. FIG. 2D shows a Taylor cone 58, liquid jet 60 and plume of highly-charged droplets 62.
The response of the analyte measured by the mass spectrometer detector increases beyond that of a capillary with no ring electrode present upon increasing the ring electrode voltage to 350 V. A Vfluid/Vring ratio of less than approximately two for a fixed distance from a counter electrode reduces the electric field at the capillary tip to the point where a stable electrospray is no longer sustainable and larger droplet formation is observed. These results indicate that an important feature of any integrated monolithic device designed for droplet formation or electrospray is control of the electric field around the orifice of a nozzle in a co- planar or near planar geometry.
The present invention provides a microchip-based electrospray device for producing reproducible, controllable and robust nanoelectrospray of a liquid sample. The electrospray device is designed to enhance the electric field emanating from a nozzle etched from a surface of a monolithic silicon substrate. This is accomplished by providing insulating layers of silicon dioxide and silicon nitride, for example, for independent application of a potential voltage to a fluid exiting at the tip of the nozzle from a potential voltage applied to the substrate sufficient to cause an electrospray of the fluid. The enhanced electric field combined with the physical asperity of the nozzle allow for the formation of an electrospray of fluids at flow rates as low as a few nanoliters per minute. The large electric field, on the order of 106 V/m or greater and generated by the potential difference between the fluid, and the substrate is thus applied directly to the fluidic cone rather than uniformly distributed in space.
To generate an electrospray, fluid may be delivered to the through-substrate channel of the electrospray device by, for example, a capillary, micropipette or microchip. The fluid is subjected to a potential voltage Vfluid via an electrode provided on the injection surface and isolated from the surrounding surface region and the substrate. A potential voltage Vsubstrate may also be applied to the silicon substrate the magnitude of which is preferably adjustable for optimization of the electrospray characteristics. The fluid flows through the channel and exits from the nozzle in the form of a Taylor cone, liquid jet, and very fine, highly charged fluidic droplets. It is the relative electric potential difference between the fluid and substrate voltages that affect the electric field. This invention provides a method of controlling the electric field at the tip of a nozzle to achieve the desired electric field for the application.
The method of fabricating an electrospray device in accordance with the present invention is also advantageous. After injection side processing is completed, the through-substrate channel is etched to a final depth, the photoresist is removed, and the substrate is subjected to an elevated temperature in an oxidizing ambient environment to grow 1-4 xcexcm of silicon dioxide on the walls of the through-substrate channel. This layer of silicon dioxide on the walls of the through-substrate channel provides an etch-stop during further processing of the substrate to define the recessed annular region. The recessed annular region may be patterned and etched from either the injection or ejection side of the substrate when the through-substrate channel is etched through the entire silicon substrate to the silicon dioxide etch stop on the ejection side of the substrate. If the through-substrate channel is not etched completely through the substrate, the recessed annular region is etched from the ejection side of the substrate. The recessed annular region may be patterned and etched to form the silicon dioxide nozzle for injection side processing or for ejection side processing.
This method does not require high alignment accuracy of features from the injection and ejection side processing to define the nozzle wall thickness thus simplifying the method. This method allows nozzles of decreasing size to be reproducibly manufactured and does not require the through-substrate channel to be etched completely through the substrate. The silicon dioxide layer that is grown on the walls of the through-substrate channel determines the wall thickness of the nozzles using this method. The desired nozzle size and use of the electrospray device determines which method is preferred. This fabrication sequence confers superior mechanical stability to the fabricated electrospray device by etching the features of the electrospray device from a monocrystalline silicon substrate without any need for assembly. Further, use of a visible alignment mark as described in the fabrication sequence of this device allows for alignment of injection side and ejection side features to better than 1 Am. This allows for overall nozzle dimensions that are smaller than previously achieved that use prior disclosed alignment schemes using infrared light. Control of the lateral extent and shape of the recessed annular region provides the ability to modify and control the electric field between the electrospray device and an extracting electrode.
This fully integrated monolithic electrospray device may be coupled with a miniaturized monolithic chromatography or other liquid sample handing devices. In particular, the electrospray device used as a means of producing a fluidic cone for spectroscopic detection including laser induced fluorescence, ultraviolet absorption, and evaporative light scattering and mass spectrometry detection. An excitation source provides a light beam. A detector detects the emission or absorbance or light scattering properties of the analytes in the fluidic Taylor cone.
The microchip-based electrospray device of the present invention provides minimal extra-column dispersion as a result of a reduction in the extra-column volume and provides efficient, reproducible, reliable and rugged formation of an electrospray. This electrospray device is perfectly suited as a means of electrospray of fluids from microchip-based separation devices. The design of this electrospray device is also robust such that the device can be readily mass-produced in a cost-effective, high-yielding process.